The present invention relates in general to methods and kits for performing nucleic acid hybridization assays and in particular to methods and kits for immobilizing a target nucleic acid on a solid support by employing a labelled nucleotide probe, a nucleotide probe attached to a first complexing agent, and a second complexing agent attached to a support.
One characteristic property of nucleic acid, which forms the heritable material of all living organisms, is its ability to form sequence-specific hydrogen bonds with a nucleic acid having a complementary sequence of nucleotides. This ability of nucleic acids to form sequence-specific hydrogen bonds (i.e., to hybridize) with complementary strands of nucleic acid has been exploited in techniques generally called hybridization assays.
In a hybridization assay, a nucleic acid having a known sequence is used as a probe to search a sample for a "target" complementary sequence. Labelling of the hybrid formed by the probe and the target permits the detection and quantitation of complementary sequence in the sample.
Because all strains of a particular microorganism share a genetic component in the form of nucleic acids susceptible to diagnosis by means of a hybridization assay, such hybridization assays are valuable research and medical tools. Detection of specific target nucleic acids enables accurate diagnosis of bacterial, fungal and viral disease states in humans, animals and plants. Additionally, the ability to probe for a specific nucleotide sequence is of potential use in the identification and diagnosis of human genetic disorders.
One approach to labelling the probe for detecting a hybrid involves binding a radioisotope (e.g. .sup.32 P or .sup.125 I) to the probe.
Non-radioactive labelling systems are also available. A first type employs a label which may be directly and covalently attached to the probe, such as fluorescent or chemiluminescent molecules (e.g., fluorescein or acridinium). A second type has a portion which is covalently attached to the DNA probe and non-covalently attached to labelled macromolecules.
An example of the second type of non-radioactive labelling system involves a biotin molecule which is covalently attached to a DNA probe and which forms a complex with fluorescent- or chemiluminescent-"labelled" avidin (or avidin derivatives such as streptavidin). Another example of the second type of non-radioactive labelling system is an antigen-"labelled" DNA probe which forms a complex with a fluorescent- or chemiluminescent-labelled antibody.
In the second type of labelling system, a probe is "labelled" with a reporter group to enable detection. A reporter is an agent which is used to associate a signal with a probe for indicating the presence or location of the probe. The signal itself., which is directly perceptible, may be generated by a separate or separable signal molecule. A label is properly a type of reporter which incorporates a signal.
Signal amplification may be achieved for biotin- or antigen-labelled DNA probes via the respective formation of a complex with avidin or with antibodies which may in turn be either covalently or non-covalently associated with an enzyme. [Leafy, et al., Proc. Natl. Acad. Sci. (USA), 80:4045-4049 (1983). This reporter group may then be incubated with the appropriate enzymatic substrate to generate a detectable signal which indicates the presence of target in the hybridization complex.
One approach to the attachment of labels to probes is described in Ward, European Patent Application No. 63,879. Ward discloses the preparation of probes having a biotin reporter molecule covalently attached to a purine or a pyrimidine ring. Selected biotinylated purines and pyrimidines are then directly incorporated within the phosphodiester backbone of nucleic acids of the probe by enzymatic means. In order to demonstrate that biotin-labelled native (double-stranded) DNA may be recognized by avidin, streptavidin or biotin-specific antibodies, Ward, et al. employ affinity chromatography. A complementary strand of DNA is synthesized on a single strand of DNA by a DNA polymerase from biotin- or iminobiotin-labelled purines or pyrimidines. The resulting, labelled, double-stranded DNA is selectively retained on an avidin- or a streptavidin-sepharose affinity column, as compared to non-labelled DNA. Ward, supra, at pages 24-26.
A biotin-labelled nucleic acid is employed in one approach to in situ hybridization in which biotin-labelled RNA is hybridized with denatured DNA in a chromosome squash. Polymethacrylate spheres are covalently attached to avidin which in turn binds to the biotin, thereby labelling portions of the DNA hybridized with the RNA. Manning, et al., Chromosoma (Berl.), 53:107-117 (1975). In addition, avidin-coated, polymethacrylate spheres have been employed in affinity chromatography to isolate biotin-labelled strands of DNA carrying a particular gene. Manning, et al., Biochemistry, 16:1364-1370 (1977).
In another approach to labelling for in situ hybridization, advantage is taken of the naturally-occurring bond between ribosomal protein and a pseudoribosomal gene in Drosophila. Antibodies are raised against the ribosomal protein and attached to polymethacrylate spheres which serve as labels for electron microscopy. Chooi, et al., Mol. Gen. Genet., 182:245-251 (1981).
The formation of a complex between an antigenic substance being assayed and one or more antibodies is also the basis for another type of biological detection technique called an immunoassay. Antibodies are white blood cell-produced proteins which are capable of combining with an antigen in a reaction which is specific for that antigen. Both antigens and antibodies may be referred to as immunological agents. An antibody only combines with certain portions (antigenic determinants) of the surface of the antigen, so that the antibody is specific to the degree that the determinant with which it combines is not also found on other antigens. At least one member of the antigen/antibody complex may be coupled to a signal molecule which permits detection, quantitative analysis on separation of the antigen/antibody complex from uncomplexed labelled antigen or antibody and other constituents of the sample. Antibodies of any type may be employed in immunoassays including polyclonal antibodies, a mixture of antibodies directed to different antigenic determinants, and monoclonal antibodies, antibodies directed to a single antigenic determinant.
Both immunoassays and hybridization techniques are employed in two-site or "sandwich" assays. In sandwich assays a target substance having the ability to form hybrid or immune complexes at two different places on the target at one time is detected.
Typically, a sandwich immunoassay involves coupling a monoclonal antibody directed to a first antigenic determinant to a solid support and exposing the support-coupled antibody to a sample containing a substance bearing the first and a second antigenic determinant. This results in the removal of the antigenic substance from the sample by the formation of a primary antibody-antigen complex which is bound to the support. Subsequent exposure of this complex to a second, labelled monoclonal antibody directed toward a second antigenic determinant on the antigenic substance creates an antibody-antigen-antibody sandwich which may be separated from the sample solution and measured. [See, e.g., David, et al., U.S. Pat. No. 4,376,110].
Sandwich hybridization assays include a two-step assay and a one-step assay. A two-step sandwich hybridization procedure involves the use of an immobilized target nucleic acid which is exposed in a first step to a first nucleic acid probe having a first portion complementary to the target and having a second portion which is not complementary to the target. In a second step, a second, labelled nucleic acid probe which is complementary to the second portion of the first probe is allowed to hybridize to the first probe, forming a "sandwich" with the first probe between the target and the second probe. Dunn, et al., Cell, 12:23-36 (1977). The sandwich hybridization procedure is relatively easy to perform and is not seriously affected by protein or other biological contaminants. Ranki, et al., Gene, 21:77-85 (1983). However, a two-step sandwich hybridization assay involves considerable delay associated with immobilization of the sample on a filter.
A one-step sandwich assay involves the use of a first nucleic acid probe immobilized on a filter. This first nucleic acid probe is complementary to a first portion of a target nucleic acid. In one step, the filter-bound first probe is exposed to a sample to be searched for the target nucleic acid sequence and to a second, labelled nucleic acid probe complementary to a second portion of the target nucleic acid, which portion is separate from (i.e., non-overlapping with) the portion of the target to which the first probe is complementary. Ranki, et al., U.S. Pat. No. 4,486,539. This one-step technique eliminates the delay caused by immobilization of a sample on a filter; eliminates differences between the types of treatment required for binding ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) to certain types of support inasmuch as the first probe may be selected to suit the support; and is even less sensitive to contaminating materials in the sample, e.g., mucus, than is a direct hybridization assay where the target is bound to the support. Ranki, et al., Curr. Top. Microbiol. Immunol., 104:307-318 (1983). Nevertheless, leakage of the first probe from the support during hybridization occurs frequently and drastically diminishes the sensitivity of the assay.
Although both immunoassays and hybridization diagnostics are more rapid than conventional tests which require viable organisms and two to three days' culture, the antigens produced in a particular disease may vary from patient to patient and from one strain of a bacterium to another or from one strain of a virus to another, so that immunological diagnosis may be difficult. On the other hand, all strains of a bacterium or of a virus share a genetic component in the form of nucleic acids susceptible to diagnosis through the use of a nucleic acid probe.
Nevertheless, it is neither easy nor convenient to attach a single-stranded nucleic acid probe directly to a solid support for use in a sandwich hybridization assay. For example, the attachment of a nucleic acid to a nitrocellulose sheet involves fixing the nucleic acid by contact with the sheet for 12-15 hours and baking the nucleic acid onto the sheet for two hours. See, e.g., Thomas, Proc. Natl. Acad. Sci. (USA), 77:5201 (1980). Such preparation of a DNA-coated nitrocellulose sheet may easily consume as much as a full working day, a factor which limits the clinical usefulness of nucleic acid hybridization.
Furthermore, because the nucleic acid probe is sequence-specific for a particular target molecule, the procedure for attaching the probe to the support must be performed for each target molecule to be detected. Thus, in order to detect a number of different DNA sequences, a diagnostic laboratory must prepare an equal number of types of supports.
In addition, it generally takes longer to hybridize complementary strands of nucleic acid than it does, for example, to form an immunological complex between an antigen and an antibody. Hybridization itself is much more quickly accomplished in solution than it is where one of the complementary sequences is attached to a solid support.
Affinity chromatographic techniques may be employed to isolate and purify nucleic acids [see, e.g., Inouye, et al., J. Biol. Chem., 23:8125-8129 (1973)] or tRNA [Miller, et al., Biochim. Biophys. Acta, 366:188-198 (1974)] or tRNA cistrons [Salomon, et al., Biochemistry, 14:4046-4050 (1975)]. However, these techniques rely upon the difficult step of forming antibodies to specific bases in a nucleic acid (Inouye, et al., supra; Salomon, et al., supra) or upon the use of a derivatized, naturally-occurring ribonucleic acid (tRNA) (Miller, et al., supra) and are thus not readily applied in general to hybridization assays.
Thus, there exists a continuing interest and need in the art for easy, convenient and rapid nucleic acid hybridization "sandwich" assays capable of accurately detecting target molecules in a sample.